RESUMO
Glucuronoxylomannan (GXM) is the principal capsular component in the Cryptococcus genus. This complex polysaccharide participates in numerous events related to the physiology and pathogenesis of Cryptococcus, which highlights the importance of establishing methods for its isolation and analysis. Conventional methods for GXM isolation have been extensively discussed in the literature. In this chapter, we describe two fast methods for obtaining extracellular fractions enriched with cryptococcal GXM.
Assuntos
Cryptococcus , Polissacarídeos , Polissacarídeos/química , Antígenos de Fungos/imunologia , Cryptococcus neoformans , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/química , HumanosRESUMO
Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to define four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifier whose overall accuracy reached 85% on the test dataset, with per-class specificity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classification. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identified and characterized using computational models so that future work may unveil morphological associations with yeast virulence.
Assuntos
Variação Anatômica , Cryptococcus/ultraestrutura , Cápsulas Fúngicas/ultraestrutura , Aprendizado de Máquina , Microscopia Eletrônica de Varredura/métodos , Cryptococcus/genética , FenótipoRESUMO
Aim:Cryptococcus neoformans is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. Materials & methods: We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Results: Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Conclusion: Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.
Assuntos
Anticorpos Antifúngicos/metabolismo , Cryptococcus neoformans/química , Polissacarídeos/fisiologia , Temperatura , Fatores de Virulência/fisiologia , Anticorpos Antifúngicos/imunologia , Cápsulas Fúngicas/química , Cápsulas Fúngicas/imunologia , Cápsulas Fúngicas/fisiologia , Pinças Ópticas , Tamanho da Partícula , Polissacarídeos/química , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Reologia , Fatores de Virulência/química , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Substâncias ViscoelásticasRESUMO
Cryptococcus neoformans is an opportunistic fungus that can cause lethal brain infections in immunosuppressed individuals. Infection usually occurs via the inhalation of a spore or desiccated yeast which can then disseminate from the lung to the brain and other tissues. Dissemination and disease is largely influence by the production of copious amounts of cryptococcal polysaccharides, both which are secreted to the extracellular environment or assembled into a thick capsule surrounding the cell body. There are two important polysaccharides: glucuronoxylomannan (GXM) and galactoxylomannan, also called as glucuronoxylomanogalactan (GXMGal or GalXM). Although GXM is more abundant, GalXM has a more potent modulatory effect. In the present study, we show that GalXM is a potent activator of murine dendritic cells, and when co-cultured with T cells, induces a Th17 cytokine response. We also demonstrated that treating mice with GalXM prior to infection with C. neoformans protects from infection, and this phenomenon is dependent on IL-6 and IL-17. These findings help us understand the immune biology of capsular polysaccharides in fungal pathogenesis.
Assuntos
Criptococose/metabolismo , Cryptococcus neoformans/fisiologia , Cápsulas Fúngicas/metabolismo , Interleucina-17/metabolismo , Polissacarídeos/farmacologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Criptococose/imunologia , Cryptococcus neoformans/metabolismo , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Interferon gama/biossíntese , Interleucina-17/biossíntese , Camundongos , Células Th17/citologia , Células Th17/efeitos dos fármacosRESUMO
The yeast-like pathogen Cryptococcus gattii is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from C. gattii, designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that C. gattii Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence in vivoIMPORTANCECryptococcus gattii has the ability to escape from the host's immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall.
Assuntos
Cryptococcus gattii/química , Cápsulas Fúngicas/química , Proteínas Fúngicas/química , Glicoproteínas de Membrana/química , Polissacarídeos/química , Fatores de Virulência/química , Animais , Linhagem Celular , Parede Celular/química , Criptococose/imunologia , Cryptococcus gattii/genética , Cryptococcus gattii/patogenicidade , Feminino , Proteínas Fúngicas/genética , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Mutação , Fagocitose , Fenótipo , Polissacarídeos/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Cryptococcus neoformans is an opportunistic fungal pathogen that is ubiquitous in the environment. It causes a deadly meningitis that is responsible for over 180,000 deaths worldwide each year, including 15% of all AIDS-related deaths. The high mortality rates for this infection, even with treatment, suggest a need for improved therapy. Unique characteristics of C. neoformans may suggest directions for drug discovery. These include features of three structures that surround the cell: the plasma membrane, the cell wall around it, and the outermost polysaccharide capsule. We review current knowledge of the fundamental biology of these fascinating structures and highlight open questions in the field, with the goal of stimulating further investigation that will advance basic knowledge and human health.
Assuntos
Cryptococcus neoformans , Cápsulas Fúngicas/metabolismo , Proteínas Fúngicas/biossíntese , Polissacarídeos/biossíntese , Parede Celular , Cryptococcus neoformans/química , Cryptococcus neoformans/citologia , Cryptococcus neoformans/patogenicidade , VirulênciaRESUMO
Cryptococcus spp. are common opportunistic fungal pathogens, particularly in HIV patients. The approved drug miltefosine (MFS) has potential as an alternative antifungal against cryptococcosis; however, the mechanism of action of MFS in Cryptococcus is poorly understood. Here, we examined the effects of MFS on C. neoformans and C. gattii yeasts (planktonic and biofilm lifestyles) to clarify its mechanism of action. MFS presented inhibitory and fungicidal effects against planktonic Cryptococcus cells, with similar activities against dispersion biofilm cells, while sessile biofilm cells were less sensitive to MFS. Interestingly, MFS had postantifungal effect on Cryptococcus, with a proliferation delay of up to 8.15 h after a short exposure to fungicidal doses. MFS at fungicidal concentrations increased the plasma membrane permeability, likely due to a direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. Moreover, MFS reduced the mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and induced DNA fragmentation and condensation, all of which are hallmarks of apoptosis. Transmission electron microscopy analysis showed that MFS-treated yeasts had a reduced mucopolysaccharide capsule (confirmed by morphometry with light microscopy), plasma membrane irregularities, mitochondrial swelling, and a less conspicuous cell wall. Our results suggest that MFS increases the plasma membrane permeability in Cryptococcus via an interaction with ergosterol and also affects the mitochondrial membrane, eventually leading to apoptosis, in line with its fungicidal activity. These findings confirm the potential of MFS as an antifungal against C. neoformans and C. gattii and warrant further studies to establish clinical protocols for MFS use against cryptococcosis.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Anfotericina B/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus gattii/metabolismo , Cryptococcus gattii/ultraestrutura , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/ultraestrutura , Fragmentação do DNA/efeitos dos fármacos , Ergosterol/metabolismo , Cápsulas Fúngicas/efeitos dos fármacos , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/ultraestrutura , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/microbiologia , Fosforilcolina/farmacologia , Plâncton/efeitos dos fármacos , Plâncton/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismoRESUMO
This study evaluated the synergistic interactions between amphotericin B (AMB) and azithromycin (AZM), daptomycin (DAP), linezolid (LNZ), minocycline (MINO), fluconazole (FLZ), flucytosine (5FC), linezolid (LZD), or tigecycline (TIG) against clinical isolates of Cryptococcus neoformans var. grubii before and after capsule induction. High synergism (>75%) was observed for the combinations, AMB+5FC, AMB+TIG, AMB+AZM, AMB+LZD and AMB+MINO but only in the strains after capsule induction. The results show that the presence of the capsule may lower the minimum inhibitory concentrations (MICs) of antifungal agents, but antimicrobial activity can be improved by combining antifungal and antibacterial agents.
Assuntos
Anfotericina B/farmacologia , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Interações Medicamentosas , Cápsulas Fúngicas/metabolismo , Criptococose/microbiologia , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/metabolismo , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects.
Assuntos
Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/fisiologia , Inibidores de Histona Desacetilases/metabolismo , Animais , Ácido Butírico/metabolismo , Divisão Celular/efeitos dos fármacos , Cryptococcus neoformans/crescimento & desenvolvimento , Modelos Animais de Doenças , Cápsulas Fúngicas/efeitos dos fármacos , Cápsulas Fúngicas/metabolismo , Ácidos Hidroxâmicos/metabolismo , Lepidópteros , Melaninas/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Fenótipo , Fosfolipases/análise , Análise de Sobrevida , Temperatura , Virulência/efeitos dos fármacosRESUMO
Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.
Assuntos
Antifúngicos/farmacologia , Criptococose/tratamento farmacológico , Cryptococcus gattii/efeitos dos fármacos , Fluconazol/farmacologia , Cápsulas Fúngicas/efeitos dos fármacos , Polissacarídeos Fúngicos/química , Animais , Criptococose/microbiologia , Criptococose/mortalidade , Criptococose/patologia , Cryptococcus gattii/química , Cryptococcus gattii/patogenicidade , Farmacorresistência Fúngica/efeitos dos fármacos , Cápsulas Fúngicas/química , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Fenótipo , Índice de Gravidade de Doença , Análise de SobrevidaRESUMO
AIMS: Glucuronoxylomannan (GXM) is the major polysaccharide component of Cryptococcus neoformans. We evaluated in this study whether GXM fractions of different molecular masses were functionally distinct. MATERIALS & METHODS: GXM samples isolated from C. neoformans cultures were fractionated to generate polysaccharide preparations differing in molecular mass. These fractions were used in experiments focused on the association of GXM with cell wall components of C. neoformans, as well as on the interaction of the polysaccharide with host cells. RESULTS & CONCLUSION: GXM fractions of variable molecular masses bound to the surface of a C. neoformans acapsular mutant in a punctate pattern that is in contrast to the usual annular pattern of surface coating observed when GXM samples containing the full molecular mass range were used. The polysaccharide samples were also significantly different in their ability to stimulate cytokine production by host cells. Our findings indicate that GXM fractions are functionally distinct depending on their mass.
Assuntos
Cryptococcus neoformans/patogenicidade , Cápsulas Fúngicas/imunologia , Polissacarídeos/imunologia , Animais , Criptococose/patologia , Cryptococcus neoformans/metabolismo , Citocinas/biossíntese , Cápsulas Fúngicas/química , Cápsulas Fúngicas/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Polissacarídeos/química , Ligação Proteica , Frações Subcelulares/químicaRESUMO
As proteínas inibidoras de poligalacturonases (PGIPs) presentes na parede celular são capazes de limitar o potencial destrutivo da poligalacturonase (PG) fúngica e, assim, constituem um tipo importante dentre os diversos sistemas de defesa do tecido vegetal frente à infecção fúngica. No mamão, o ataque fitopatogênico é o principal causador de danos pós-colheita, e sua alta susceptibilidade pode estar relacionada com a baixa eficácia ou pouca abundância dos meios de defesa anti-fitopatogênica. Uma vez que isso pode estar relacionado com as PGIPs e nada se conhece sobre o papel dessas proteínas nesse fruto, o objetivo do trabalho foi clonar os genes das PGIPs de mamoeiro e definir seu padrão de expressão em diferentes órgãos e tecidos e ao longo do amadurecimento. Para tanto, foram identificadas no genoma do mamoeiro, a partir de critérios que definem a identidade de uma PGIP, duas prováveis sequências dentre 13 candidatas iniciais. Ambas foram clonadas a partir das sequências genômicas e de cDNA, sequenciadas e sua identidade confirmada, sendo denominadas Cppgip4 e Cppgip6. As análises de expressão relativa em diversos tecidos e idades fisiológicas do mamoeiro demonstraram que os dois genes apresentaram diminuição da expressão com o desenvolvimento dos frutos, sendo que com a polpa apresentou redução dos níveis de expressão relativa de Cppgip4 em até 18 vezes dos 30 dias pós-antese (DPA) ao 9 dias pós-colheita (DPC). Na casca também houve redução significativa da expressão com o desenvolvimento. Para a expressão absoluta, nos frutos, sementes, caules, raízes e folhas, o número de cópias de ambos os transcritos decresceu com o desenvolvimento, sendo cerca de cem mil vezes mais abundante para Cppgip6 que para Cppgip4. As tentativas de expressão de proteínas recombinantes em Pichia pastoris não geraram resultado positivo, provavelmente em virtude das condições ideais de indução ainda não terem sido estabelecidas corretamente para o ensaio. A atividade de PGIPs extraídas diretamente do tecido foi medida por análise de difusão em ágar empregando pectinase de Aspergillus niger e revelou uma tendência à diminuição da porcentagem de inibição à medida que os frutos se desenvolveram, em concordância com os resultados da análise por qPCR. O conjunto de resultados sugere que a expressão varia com o estádio de desenvolvimento do fruto e é tecido-específica, possivelmente em resposta à diferente susceptibilidade dos tecidos ao ataque fitopatogênico, indicando que menores níveis de transcritos e atividade no amadurecimento, período de maior susceptibilidade, poderiam sinalizar para a regulação do processo degradativo marcando o início da senescência
Polygalacturonase inhibiting proteins (PGIPs) present in plant cell walls are able to inhibit the destructive action of fungal polygalacturonase (PG). In this way, they constitute an important type of plant defense system against fungal infections. In papaya fruit, the pathogenic attack is the main cause of post harvesting loss, and its high susceptibility may be related to the low efficiency or low abundance of anti-phytopathogenic defense. Since this fact could be related to PGIPs expression and little is known about the response of these proteins in the fruit, the aim of the present work was to clone the genes of PGIPs papaya fruit and set their expression pattern in different organs and tissues throughout fruit ripening. Thus, two probable PGIP sequences among 13 initial candidates were identified in the papaya genome by using specific criteria. Both sequences were cloned from cDNA and genomic samples, sequenced and confirmed its identity, and then being named Cppgip4 and Cppgip6. Analysis of relative expression in various tissues at different physiological stages demonstrated that both genes were down regulated during fruit development. The relative expression levels of Cppgip4 in papaya pulp was reduced by 18 times from the 30 days post-anthesis (DPA) to the 9 days post-harvest (DPH). Similarly, gene expression in papaya peel was significant down regulated during fruit development. Absolute expression analysis revealed gene expressions in the fruit pulp, seed, stem, root and leaf were also down regulated within development. Moreover, Cppgip6 gene expression was a hundred thousand times more abundant than Cppgip4. The recombinant protein expression in Pichia pastoris did not result positive, probably because of the ideal conditions of induction have not been properly established the yet. The activity of PGIPs extracted directly from the tissue was measured by the agar diffusion assay using pectinase from Aspergillus niger and showed decrease of inhibition during fruit developed in accordance with the results of the qPCR analysis. Based on the results it is possible to suggest the expression of these genes varies temporally with the developmental stage of the fruit and is tissue-specific, possibly in response to the different susceptibility of tissues to pathogenic attack. In addition, the lowest levels of PGIP expression were achieved at the fruit ripening, when the susceptibility to fungal infection is high and could signal for regulating the degradation process characterized by the onset of senescence
Assuntos
Poligalacturonase , Poligalacturonase/análise , Testes de Sensibilidade Microbiana/instrumentação , Clonagem de Organismos/métodos , Carica/classificação , Pichia , Aspergillus niger , Expressão Gênica , Cápsulas Fúngicas , Infecções , Biologia Molecular/métodosRESUMO
The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.
Assuntos
Cryptococcus neoformans/patogenicidade , Cápsulas Fúngicas/metabolismo , Fagocitose/efeitos dos fármacos , Polissacarídeos/metabolismo , Aglutininas do Germe de Trigo/farmacologia , Animais , Encéfalo/microbiologia , Quitina/metabolismo , Criptococose/tratamento farmacológico , Criptococose/patologia , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/metabolismo , Cápsulas Fúngicas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Aglutininas do Germe de Trigo/metabolismoRESUMO
In the present study, the in vitro susceptibility and capsular width from both melanized and non-melanized Cryptococcus neoformans cells in the presence of Pimenta pseudocaryophyllus crude extract were determined. The results were compared with those obtained for voriconazole and amphotericin B. Melanization was obtained in minimal medium broth with the addition of L-dopa, and the antifungal susceptibility tests were performed using the broth microdilution method. Capsular width of 30 cells of each one of the isolates in medium with crude extracts of P. pseudocaryophyllus or voriconazole or amphotericin B at a concentration corresponding to 0.5 times the minimal inhibitory concentration (MIC) was measured, and the mean was calculated. The MICs and minimal fungicidal concentrations (MFCs) for plant extract and voriconazole were identical for both melanized and non-melanized C. neoformans isolates, but for amphotericin, the MFCs for melanized cells were up to 8 times higher than for non-melanized cells. The capsular width of C. neoformans cells was smaller (p < 0.001) in the presence crude extract of P. pseudocaryophyllus and of voriconazole regardless melanization. The findings of capsule alterations of C. neoformans verified in this study provide fertile ways for future research into the effects of antifungal agents on the pathogenesis of cryptococcosis.
Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Melaninas/metabolismo , Pimenta/química , Extratos Vegetais/farmacologia , Anfotericina B/farmacologia , Criptococose/microbiologia , Cryptococcus neoformans/química , Cryptococcus neoformans/metabolismo , Farmacorresistência Fúngica , Cápsulas Fúngicas/química , Cápsulas Fúngicas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Pirimidinas/farmacologia , Triazóis/farmacologia , VoriconazolRESUMO
The capacity of the 4B3 monoclonal antibody, previously obtained by immunization of BALB/c mice with the capsular polysaccharide of Cryptococcus neoformans was studied to recognize this structure as part of the intact cell of this yeast. With this aim, 4B3 was evaluated by cellular ELISA and indirect immunofluorescence, using as an antigen a cell suspension of the 028 LMIPK C. neoformans strain. Serum from the mouse used in the fusion for obtaining 4B3 monoclonal antibody was used as a positive control, whereas an anti-dengue monoclonal antibody was used as a negative control. Both methods demonstrated that the above mentioned monoclonal antibody is capable of recognizing the native antigen, which will allow its future evaluation for diagnostic and therapeutic purposes.